Iroa metabolomics workflow for improved accuracy, identification and quantitation

ABSTRACT

An IROA Matrix of metabolite compounds is disclosed. Each of whose compounds has a molecular weight of 2000 AMU or less, and is present as first and second isotopomers that are equally present at two predetermined isotopomeric balances, and contain 2 to 10% of a first isotope, and 90 to 98% of a second isotope, respectively. A reagent pair for transforming a natural abundance mass spectral analysis metabolite sample into an IROA sample is also disclosed and comprises two reactively identical reagents that constitute first and second isotopomers containing 2 to 10% of a first isotope, and 90 to 98% of a second isotope, respectively. Each of the reagent pair contains the same reactive group that reacts with and bonds to a functional group of one or more compounds present in a composition of biologically-produced metabolite compounds. Methods of making and using the above and related materials are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims benefit of Provisional Patent application No.62/463,153, entitled “Implementing IMS-assisted IROA for Metabolomics”filed on Feb. 24, 2017, whose disclosures are incorporated by reference.

BACKGROUND ART

Metabolites are small molecular weight compounds (less than about 2000Da and more usually less than about 1000 Da) that are employed asbuilding blocks or produced as end products in various metabolicpathways and cellular regulatory processes in a biological system. Theentire collection of metabolites in a biological system, whether at thecellular, pathway or organism level, is known as a “metabolome”. Levelsof these metabolites in a metabolome are either dictated by the genome,proteome, and/or transcriptome of the biological system or imposed byenvironmental perturbations and results in changes in phenotype. Thus,metabolomics can be applied to map or identify the cause of alterationin phenotype and understand correlations between “omics”. Dwivedi, etal., Int J Mass Spectrom 2010 298:78-90.

Isotopic Ratio Outlier Analysis (IROA) has been developed to enable thecharacterization of carbon information in a given metabolites or afragment. Unlike other stable isotope labeling methods, rather thanutilizing substrates with natural abundance (1.1% of 13C isotopomer seenin carbon atoms in nature) and 98-99% enrichment for the control andexperimental populations, respectively, IROA with prototrophic yeastuses randomized 95% 12C glucose (5% 13C), and 95% randomized 13C glucose(5% 12C) as carbon sources. This strategy leads to more predictable anddiagnostic patterns for the observable isotopic peaks in the massspectra. [Qiu et al., Metabolites 2018 8:9].

The promise of IROA for metabolic phenotyping has been demonstrated inmodel organism studies. Saccharomyces cerevisiae, a prototrophicwild-type strain in the CEN.PK background [Brauer et al., Mol. Biol.Cell 2005, 16:2503-2517] was grown in minimal yeast nitrogen base (YNB)media, containing either randomized 95% 12C, or 95% 13C glucose as themain carbon source, in order that the isotopomer pattern of allmetabolites would mirror the labeled glucose [Qiu et al., Anal. Chem.2016, 88:2747-2754], a protocol that can easily be adapted for microbialspecies studies.

The abundance of the light isotopologues in the 95% 13C samples(M_(n−1), M_(n−2), etc., the 13C envelope) or the heavy isotopologues inthe 95% 12C samples (M₀₊₁, M₀₊₂, etc., the 12C envelope), follows thebinomial distribution for 13C, based on the initial substrateenrichment, in the metabolite products generated. The mass differencebetween the 12C (M₀) isotopic peak and the 13C (M_(n)) isotopic peakindicates the number of carbons (n) in the metabolite's carbon backbone.This narrows possibilities for chemical formula generation (CFG) and fornormalization between control (13C) and treated (12C) groups. [Qiu etal., Metabolites 2018 8:9].

It is possible to use metabolomic techniques, such as the IROA basic, orIROA phenotypic protocols (optimally)[de Jong and Beecher, C.Bioanalysis 2012, 4 (18):2303-2314], or standard metabolomic techniquesto identify and crudely quantify several hundred or even thousands ofcompounds in a biological sample. However, to make such measurements andto compare the measurements from any two or more samples, all thesamples need to be analyzed in a single batch, ideally during a singleday because day-to-day variances are too great to otherwise overcome,and absolute quantitation; i.e., relative to a known standard, cannot beassured.

It is currently not quantitatively acceptable to compare samples run onthe same instrumentation several days apart, and impossible to comparedata generated on different instruments, or based on different methods.Instrument drift, chromatographic drift, and even environmentalconditions can alter results sufficiently so that reproducibility ishard to obtain even on the same instrument. In addition to theseproblems of quantitation, the identification of any compound across manymass spectral techniques alone is unlikely to be successful unless verycareful calibrations have been made and authentic standards are run.This is because, not only are there multiple biological compounds thatcan be confused because they have the same exact mass but, even moreproblematic, there are often more artefactual or fragmentary compoundsthat are structurally different from, but can share the correct mass, oreven formulae, as biological isobaric equivalents.

The invention disclosed hereinafter extends methods described in thefollowing U.S. Pat. No. 7,820,963, the basic IROA patent, issued Oct.26, 2010, referred to hereinafter as IROA963; U.S. Pat. No. 7,820,964,issued Oct. 26, 2010, and referred to hereinafter as IROA964; U.S. Pat.No. 8,168,945, issued May 1, 2012, referred to hereinafter as IROA945;U.S. Pat. No. 8,536,520, issued Sep. 17, 2013, referred to hereinafteras IROA520; and U.S. Pat. No. 8,969,251 that issued Mar. 3, 2015, and isreferred to hereinafter as IROA251. These patents and the art citedtherein are incorporated herein by reference.

The IROA protocols rely on the creation of isotopic patterns that aremathematically informative to insert information into biological samplesto provide better identification and quantitation of the individualchemical components when the samples are subjected to mass spectralanalysis. Traditional methods required chromatographically clean; i.e.,“baseline”, separation to achieve the best quantitative accuracy, theIROA protocols do not and hence can be used in the quantitation of verychemically complex samples where such separation is not consistentlypossible.

The exemplary samples studied were uniformly and universally labeledwith appropriate isotopes. An element in which there are two stableisotopes that are not significantly distinguished by enzymes or livingsystems is preferably used. Carbon (specifically, 12 C and 13C) is usedfor purposes of illustration herein because of its universalapplicability. However, additional examples are well known to a workerof ordinary skill.

The use of isotopes that exhibit minimal biological isotope effect is ofimport. For instance, the use of the isotopes of hydrogen such asdeuterium (D) is not suitable because it frequently causes an observableeffect on metabolism due to the fact that the deuterium isotope has amass that is twice that of hydrogen, and thus causes a reduction in thekinetics of some enzyme mechanisms. Tritium (T) is radioactive and thusnot stable to decay.

In many of these protocols the production of the IROA patterns relies onthe creation of molecules where the probability of all carbons in amolecule is carefully constrained to a close range of isotopicprobabilities. Illustratively, for a system using stable isotopes ofcarbon [carbon-12 (12C) and carbon-13 (13C)], the isotopic ratios inthis example specifically include a dilution of five to ten percent ofone carbon isotope in another; i.e., one sample is grown on a carbonsource (nutrient in a medium) that can be 95% carbon-12 (12C) and 5%carbon-13 (13C), hereinafter called “C-12 medium”, and in such asituation the other sample is grown in mirrored medium that contains anutrient that contains 95% carbon-13 and 5% carbon-12 in a medium,hereinafter called “C-13 medium”. In each of these cases the biologicalsystem takes up the nutrient in the medium and grows upon it in such away as to transform itself so that all of its parts are distinctivelyidentifiable as to their origin. Further information can sometimes beobtained by incorporating a second set of two isotopes of a second atompresent at two different predetermined isotopic ratios into the nutrientcompositions.

When the two samples are mixed, intermingled or otherwise composited,the composite sample contains molecules from both the “control” (thatare made up of a substantial majority, e.g., 90% to 95%, of 12C) and the“experimental” (that are made up of a substantial majority, e.g., 90% to95%, of 13C). Deviating significantly from the 90% to 95% ratio taughtby this method reduces the potential for interpretation as is taught inIROA963, although 98% and 2% of the carbon isotopes have beensuccessfully used.

More specifically still, the probability can be set to 95% C-13 in anillustrative IROA standard sample. In such a standard all the moleculescontained in it exhibit the property that the probability for of itscarbons will be as close to 95% 13C as is achievable. Such IROAmolecules have many special properties, namely:

1) The isotopic balance of 12C to 13C is so much larger than the naturalabundance probability of approximately 1.1% and yet is specifically notapproaching 100%, therefore each molecule presents itself as acollection of isotopomeric sets of that molecule with the mass of eachset differing by the mass of exactly one carbon neutron, orapproximately 1.00335 AMU. These sets are significantly larger and morecomplex than natural abundance equivalents and can be easily identified.

2) The distribution of isotopomers across the above sets is a functionof the number of carbons in the molecule and the probability of a 13C ineach such position. The presence of isotopomeric sets contributed fromother natural abundance sources of hydrogen, oxygen, nitrogen, etc. areso small that their patterns are equally distributed into andinsignificant to the C13 isotopomeric sets.

3) The amount of isotopomers for each IROA molecule can be deduced in amass spectrometer as the height of a peak, and therefore the relativeconcentration of all isotopomeric sets creates a pattern of peaks foreach molecule. This pattern is effectively defined as a binomialdistribution the percent (x), and the number of carbons (n), andtherefore can be calculated as probabilities, ((1−x)+(x))n.

4) These IROA patterns are dominant features of any mass spectralanalysis of an IROA sample. Because the patterns themselves can be quitecomplex, their occurrence due to random peak noise is effectivelynon-existent. Software was developed that identifies these patterns withgreat accuracy.

5) The C12 and C13 monoisotopic masses of such a molecule cannot be seenbut can be determined by inspection of the shape of the patterns seen.The monoisotopic mass constrained by the number of carbons effectivelyis an unique determinant of the molecular formula of the molecule,significantly more accurate than attempting to solve the polynomialequations required for natural abundance molecules.

6) Aside from the mass differences of their isotopomeric sets, themolecules are otherwise indistinguishable and thus perform verysimilarly through almost all treatments and generally have the samephysical characteristics. This characteristic of IROA peaks is a basisof the IROA Identification Techniques.

There are many IROA protocols based on these properties. The followingtwo IROA protocols are relevant to this invention.

The Basic IROA Protocol

The basic IROA protocol (which was described in IROA963, and continuedin IROA945, and IROA520) creates two populations of IROA moleculescontaining widely different amounts, typically 90-95% and 10-5% of thefirst and second isotopes, respectively, and 10-5% of the first isotopewith 90-95% of the second isotope. Isotopes other than hydrogen anddeuterium are preferred such as the particularly preferred approximately5% C13 and approximately 95% C12 used with approximately 5% C12 andapproximately 95% C13.

In both populations, the distribution of C13 in every compound is randomand universal and the probability of a carbon being either a C12 or aC13 is the stated value, here either approximately 5% or 95%. Theexperimental “base” population of molecules (C12-B) with approximately5% C13 and the remaining carbons (95%) are C12. The control “InternalStandard” population (C13-IS) sample made up of approximately 95% C13and 5% C12.

Because both the C12-B and C13-IS are made up of IROA molecules:

1) For any given molecule their respective peak patterns are different,but both solve to the same molecular formula. The C12-B monoisotopicpeak has a distinct M+1, M+2 peaks, and possibly additional M+n peaks.The C13-IS monoisotopic peak has a distinct M−1, M−2 peaks, and likelyadditional M−n peaks.

2) Unlike isotopomers based on deuterium, these isotopomersco-chromatograph and exhibit very similar physical properties except formass.

3) IROA compound peaks can only be created in most experimental systemsthrough biological means (IROA520), but intentional synthetic IROAcompounds (IROA964) can also be prepared and added. In this workflow,the presence of an IROA signal assures that all IROA patterns can comeonly from the C13-IS or the C12-B, and that they are immediatelydistinguishable from artefact, electronic noise, or any spurious signalsthat are always be based on natural abundance isotopic signatures.

4) When the patterns from the same molecule from both the C12-B sampleand the C13-IS are found in the same sample, the paired signal is atriply redundant information system in which:

-   -   a) the number of carbons in the molecule can be determined by        the ratio of the height of the M+1 to the C12-B monoisotopic for        the IROA molecules coming from the experimental samples,    -   b) the number of carbons in the molecule can be determined by        the ratio of the height of the M−1 to the C13-IS monoisotopic        peak for the IROA molecules coming from the C13-IS samples, and    -   c) the number of carbons in the molecule can be determined by        the mass difference between the monoisotopic mass of the        molecules coming from the experimental sample, and the mass of        the monoisotopic from the C13-IS.

When all three of these calculations indicate the same number ofcarbons, it is extremely likely that the pattern has been correctlyfound, and that the probability of error is extremely low. Becausediscovery of these patterns can be entirely software-driven, thediscovery of such peaks is a completely automatable task (IROA945).

The basic IROA protocol permits for a completely unbiased (ornon-targeted) analysis of an experimental sample in which the C12-B canbe made to vary according to an experimental design for purposes ofdiscovery of the biological effect of such experimental design. In sucha sample the C12-B population is derived from an experimental sample,and if a molecule does not happen to be in either the C13-IS or theC12-B sample, the presence and probable identification of the moleculeis still possible, and the absence of the molecule in the other is anestablishable fact.

Although not triply redundant, the presence of a randomly created (i.e.artefactual) IROA peak is so low that a single IROA peak is easilyidentified as such and can be quantified. This basic IROA protocol istherefore suited to experimental situations in which the ability to findand characterize all the peaks of biological origin in either the C12-B1or C13-IS, thereby identifying those situations in which a molecule ispresent in one but missing from the other.

The triple redundancy of the basic IROA protocol is such a strongalgorithm that it is possible to find very weak signals even in thepresence of very strong noise by simply enforcing the peak shaperequirements.

In the case of Matrix, where the C12-B and C13-IS sides are both ofequal chemical composition and matching isotopic balance, by design, therequirement for symmetry makes it easy to find many very small peaks indeep noise situations with little chance of error. Thus, Matrixrepresents a special case of the IROA Basic protocol in which itscharacteristics are so predictable as to make the information derivedfrom it especially reproducible and capable of being found at extremelylow levels of detection.

The IROA Workflow is based on this unique property. The source ofmaterial for Matrix can be either biological or synthetic. The IROAIdentification Techniques can be applied to any IROA peak to furtherstrengthen the identification of the underlying compound.

The Phenotypic IROA Protocol

The Phenotypic IROA protocol is a protocol for situations in which it isnot feasible or practical to label the experimental sample itself but acommon and consistent 95% (+/−3%) IROA internal standard, such as theabove described C13-IS, is used to assure accurate identification of amolecule and accurate quantitation. The Phenotypic Protocol is usefulfor the analysis of human (clinical) samples, agricultural samples,industrial samples, or other situations where the size or the source ofthe experimental samples is such that it is simply not feasible to labelthem. However, the Phenotypic protocol, by providing a common rigorousIROA internal standard, provides a more accurate route for theidentification and quantification of a large number of compounds thatare found in the sample natural abundance isolates.

Unlike the “unbiased” or “non-targeted” analysis of basic IROA,Phenotypic IROA is a targeted quantitative analysis of a very largenumber of compounds based on a very chemically complex IROA internalstandard (IS). A C13-IS can contain well over 1000 compounds(potentially unlimited), but the IROA properties outlined earlier do notrequire complete chromatographic separation to assure both the identityand quantitation of all the compounds contained in the IS.

The Phenotypic protocol puts an IROA internal standard into everynatural abundance sample and uses the dual pieces of information fromthe C13-IS, 13C-monoisotopic mass and number of carbons, to locate thenatural-abundance isotopomer of the same compound. Correlation of thenatural abundance time-resolved chromatographic profile of the foundpeak, and it's natural-abundance isotopic form are then used to supportthe IROA-based identification.

Because the IROA peaks are informatically self-contained, it is possibleto correctly identify and quantify multiple co-eluting peaks. In thecase of the Phenotypic Protocol, the IS can be created by a worker toprovide support for the unique quantitation needs of the experimentalsystem. Thus, a wheat researcher, can create a wheat C13-IS that can beused because it contains a chemical profile more reflective of wheatbiochemistry, but this C13-IS is used primarily to find and identifyIROA peaks in wheat and quantify their natural abundance counterparts.Although the triple redundancy of the Basic IROA protocol does not existin the Phenotypic protocol, the signal is still redundant in that the95% C13-IS provides a mass and number of carbons to determine exactlywhere the natural abundance monoisotopic signal is found (see FIG. 10F).

In the IROA workflow, the same C13-IS is used in both the Matrix and theClinical or experimental samples and the chemical information derivedfrom the Matrix sample is used to verify and validate the compoundsfound in the clinical or experimental (Phenotypic) samples. ThePhenotypic samples can be analyzed for chemical information to the sameextent as the Matrix samples but this is not required. For instance,whereas the Matrix samples need to be analyzed to completelycharacterize every compound present in it, it can be sufficient to usethe mass and retention information derived from the analysis of theMatrix to find the same compounds in the experimental or clinicalsamples, and use a higher acquisition rate than would be possible in theMatrix samples to achieve a higher quantitative accuracy.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention contemplates an IROA Matrixcomposition of biologically-produced metabolite compounds. Each of thosemetabolite compounds has a molecular weight of about 2000 AMU or less.Each of the metabolite compounds is present as first and secondisotopomers that are equally present at two predetermined isotopomericbalances. The first isotopomers contain about 2 to about 10% of a firstisotope, and the second isotopomers contain about 90 to about 98% of asecond isotope of the same atom. The first and second isotopes arestable to radioactive decay and are other than hydrogen and deuterium.

The biologically-produced metabolite compounds are obtained from a celllysate preparation obtained from culture of single-celled ormulti-celled organisms, and the molecules are randomly and universallylabeled with isotope pairs of one or more elements selected from thegroup consisting of isotopes of carbon (12C and 13C), nitrogen (14N and15N), oxygen (16O, 17O, or 18O), sulfur (32S, 33S, 34S, or 36S),chlorine (35Cl and 37Cl), magnesium (24Mg, 25Mg and 26Mg), silicon(27Si, 28Si and 29Si), calcium (40Ca, 42Ca, 43Ca, and 44Ca), and bromine(79Br and 81Br).

Another contemplated aspect of the invention is a method of creating areference library of identity data of compounds in an IROA Matrix asdescribed above, and comprises the steps of 1) mass spectrallydetermining the identity of the compounds of an IROA Matrix that arewithin the resolution and sensitivity of the apparatus to provide itssymmetrical IROA peak pattern, and additionally determining one or moreof: a) the gas and/or liquid chromatographic properties of the compoundspresent, b) the collisional cross section of the compounds present, andc) the fragmentation pattern of the compounds present. The compoundidentity data so determined is maintained for use in identifying one ormore of the same compounds in a later-analyzed sample. The referencelibrary of identity data of compounds in an IROA Matrix is itself alsocontemplated. The use of one or both of compound collisional crosssections and fragmentation patterns are preferred in conjunction withmass spectral identification.

A further contemplated invention is a method of quantifying andidentifying compounds in a natural abundance sample using an InternalStandard that is of the same chemical composition as isotopomerscontaining the about 90 to about 98% of the heavier molecular weightisotope-containing compounds of an IROA Matrix composition and isinserted into that natural abundance sample. Each compound in theInternal Standard is itself identified in a before-described referencelibrary of identity data. It is preferred that the quantity of eachidentifiable compound of the natural abundance sample is determined, andmore preferably, the quantity of each natural abundance sample compoundis determined relative to the Internal Standard.

Yet another aspect of the invention is a method of measuring qualityassurance and/or a quality control on the operational constancy of amass spectral apparatus and associated ion mobility channel andchromatographic apparatus, when present. That method comprises the stepsof assaying the sample of an IROA Matrix composition as described above,and determining whether the same sets and amplitudes of symmetric IROAmass spectral peaks are present in each analysis. The preferences notedabove in regard to an IROA Matrix composition are repeated here and ineach time a Matrix composition or its components are used herein.

A still further aspect of the present invention contemplates a reagentpair capable of transforming a natural abundance mass spectral analysismetabolite sample into an IROA sample. That reagent pair comprises tworeactively identical reagents that constitute first and secondisotopomers. The first isotopomers contain about 2 to about 10% of afirst isotope, and the second isotopomers contain about 90 to about 98%of a second isotope of the same atom. The first and second isotopes arestable to radioactive decay and are other than hydrogen and deuterium.Each of the reagent pair contains the same reactive group that reactswith and bonds to a functional group of one or more compounds present ina composition of biologically-produced metabolite compounds. Each of thebiologically-produced metabolite compounds of the natural abundance massspectral analysis sample has a molecular weight of about 2000 AMU orless.

A reagent pair reactive group reacts with and bonds to abiologically-produced metabolite functional group selected from thegroup consisting of one or more of an amine, aldehyde or ketone,hydroxyl, thiol and carboxylic acid. Preferably, a reactive group reactswith and bonds to an amine functional group. A preferred reactive groupis a isothiocyanate reactive group, and the reagent pair are isotopomersof phenylisothiocyanate whose first isotopomers contain about 2 to about10% of a first isotope, and whose second isotopomers contain about 90 toabout 98% of a second isotope. An alternative pair of reagents are IROAisotopomers of a hydrazine or a semicarbazide that react and bind tocarbonyl groups of aldehyde and ketone groups present in a naturalabundance mass spectral analysis metabolite sample.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings forming a portion of this disclosure,

FIG. 1A shows the mass spectral peaks obtained on the analysis oflactose that contains naturally abundant amounts of 12C and 13C, FIG. 1Bshows the mass spectral peaks obtained on the analysis of lactose thatcontains 95% 12C and 5% 13C, FIG. 1C shows the mass spectral peaksobtained on the analysis of lactose that contains 5% 12C and 95% 13C,and FIG. 1D shows the mass spectral peaks obtained on the analysis oflactose that contains equal amounts of lactose that contains 5% 13C with95% 12C along with 95% 13C and 5% 12C;

FIG. 2 illustrates the spectra of lactose containing natural abundanceof both 12C and 13C as well as peaks obtained from lactose containing95% 13C, and illustrates the number of carbon atoms in the assayedmolecule by the difference in m/z value of the two base peaks being 12AMU;

FIG. 3 illustrates the symmetrical arrangement of IROA peaks with thedifference between the m/z values for the two base peaks defining thenumber of carbon atoms present in the assayed compound. See, e.g., deJong, F. A.; Beecher, C. Bioanalysis 2012, 4 (18), 2303-2314;

FIG. 4 broadly illustrates steps in the preparation of an IROA sampleprepared separately from Saccharomyces cerevisiae grown in a medium thatcontains 5% 13C or 95% 13C as the main source from which asolvent-soluble (usually water) cell lysate is prepared, providing twosolutions that contain the same amount of each yeast metabolite compoundpresent and containing either 5% 13C or 95% 13C, which are then combinedto form a pooled extract that is freeze-dried to form a reconstitutableIROA standard referred to herein as “Matrix”. See, e.g., Qiu, et al., J.Anal. Chem. 2016, 88 (5), 2747-2754;

FIG. 5 whose upper portion shows a schematic of a IM apparatus drifttube with the analyte ions within the dashed rectangle and ions ofunknown origin outside of that dashed rectangle and a simulated ionmobility spectrometric- (IMS-) Assisted IROA mass spectrum containingpeaks with added X's above them to indicate the peaks due to those ionsof unknown origin, and in which ions of unknown origin that interferewith detected IROA masses but have been separated by ion mobility arerepresented with black circles;

FIG. 6 is similar to FIG. 5, except that this figure is a simulation inwhich two isomers are detected and as seen in the upper schematic IMdrift tubes whose separated analytes are shown within the two dashedrectangles and ions of unknown origin outside of those dashed rectangleswith the same meanings as in FIG. 5, and because there are IROA internalstandards for them, they can be independently quantitated, which wouldnot be possible without both the IROA internal standards and ionmobility. See, e.g., Dwivedi et al., Int. Jounal Mass Spectrom. 2010,298 (1-3):78-90 that discusses use of IMS-assisted mass spectroscopy;

FIG. 7A illustrates a LC-IM-MS analysis of a portion of the pooled yeastextract (IROA Matrix) prepared as discussed in FIG. 4 and in greaterdetail hereinafter, in which the portion of the LC separation within thebox is the portion analyzed mass spectrally and the line beneath theboxed line illustrates the elution solvent gradient, FIG. 7B illustratesa portion of the LC separation that was analyzed (boxed peak) with theresulting mass spectrum for an 11 carbon compound adjacent to the LCtrace, and FIG. 7C illustrates further details of the separation and MSanalysis in the upper portion such as retention time, mass range anddrift time, as well as IM analysis readily showing the IROA peakpattern, specific drift time and number of carbons in the analyzedcompound;

FIG. 8A illustrates a LC-IM-MS analysis of another peak (boxed) from aLC separation in which a 5 carbon compound was the analyte, and FIG. 8Billustrates one IROA recognized pattern using LC-MS separation withfurther details that indicate that two compounds are present from the IMdata;

FIG. 9A illustrates an overlapping MS peak pattern in compounds obtainedfrom the boxed LC peak on the left side of the figure, FIG. 9Billustrates the deconvoluted spectra in which the IM data indicate thattwo nine carbon compounds have the same chemical formula, and drifttimes matched with identity data reference libraries and that a tencarbon compound was also present, and FIG. 9C shows the mass spectra foreach of the three compounds identified;

FIG. 10A is a mass spectrum of arginine in which C12 and C13 are presentin natural abundance, whereas FIG. 10B shows the similar spectrum usingarginine that contains 95% C12 and 5% C13, FIG. 10C shows the massspectrum for arginine that contains 95% C13 and 5% C12, FIG. 10D showsthe basic IROA spectrum of arginine when equal amounts of the compoundcontaining 95% C12 and 5% C13 and the compound 95% C13 and 5% C12 arepresent, FIG. 10E illustrates triply redundant IROA peak patterns forarginine natural abundance noise in which relationship between themonoisotopic peaks is assured when the height of the C12 M+1, the C13M−1, and the mass difference between the two monoisotopic peaks allindicate the same number of carbons are present in the molecule, andFIG. 10F illustrates Phenotypic “redundancy”, in which the identity ofthe natural abundance peak is confirmed by both the molecular formula ofthe 13C monoisotopic peak and the number of carbons indicated by theheight of it's M−1 provided by admixture of the C13-IS sample to thesample for analysis. Peaks associated with arginine are starred (*) ineach spectrum of FIGS. 10A-D;

FIG. 11A shows mass spectral peaks present when adenosine is assayedusing a sample containing equal amounts equal amounts of the compoundcontaining 95% C12 and 5% C13 and the compound 95% C13 and 5% C12, andFIG. 11B shows mass spectral peaks present when phenylalanine is assayedusing a sample containing equal amounts equal amounts of the compoundcontaining 95% C12 and 5% C13 and the compound 95% C13 and 5% C12. It isnoted that the number of carbon atoms present is provided by thedifference in m/z values for the base peaks in each spectrum;

FIG. 12A illustrates the mass spectral IROA peak pattern forphenylalanine based on use of a mixture of equal amounts of 5% C12 and95% C13 phenylalanine and 95% C12 and 5% C13 phenylalanine; FIG. 12Billustrates the mass spectral IROA peak pattern for that samephenylalanine sample after SWATH fragmentation; and FIG. 12C providesIROA diagnostic structural information via fragment interpretations fromthe peaks of FIG. 12B;

FIG. 13A and FIG. 13B illustrate that derivatized peaks of argininemaintain their IROA character in ion mobility [with and withoutdifferential mobility spectrometry (DMS)] when derivatized using eitherisotopically labeled IROA compounds (FIG. 13A) or with natural abundancecompounds derivatized with isotopically labeled reagent such as a 95%C13 phenylthiocarbamyl (PITC) group (FIG. 13B). The collection ofisotopomers appear as a unit in Ion Mobility, here Sciex SelexIon™.

FIG. 14A and FIG. 14B illustrate a similar maintenance of IROA characterfor similarly prepared and assayed tyrosine derivatives;

FIG. 15A, FIG. 15B, FIG. 15C and FIG. 15D illustrate the power of IROA,particularly in conjunction with ion mobility to separate complexspectra into their component individual spectra. Thus, the isotopomericcollections of IROA peaks remain IROA peaks in IM, here using an Agilent6560 (ion mobility time of flight) IM-QTOF machine, that uses Drift TubeIM (DT-IM). Although the ClusterFinder™ software separates outoverlapping IROA peaks based on mass differences in the pre-IM Massspectrum (FIG. 15A) here two co-eluting IROA peaks are separated cleanlyin the IM (FIG. 15B) for complete compound spectral identification (FIG.15C and FIG. 15D) based on their IROA characteristics.

DEFINITIONS

As used herein, the abbreviations “13C”, “C13” and “¹³C” all refer tothe isotope of the element carbon that has an atomic weight of 13 AMU.Similarly, the abbreviations “12C”, “C12” and “¹²C” all refer to theisotope of the element carbon that has an atomic weight of 12 AMU.

Chromatography can mean any form of a chemical separation, including butnot limited to all forms of liquid chromatography (LC), gaschromatography (GC), capillary electrophoresis (CE), ion mobility (IM),solid phase extraction (SPE), etc.

Compound identification means any method of determining the physicalcharacteristics of a chemical compound, including but not limited tomass spectroscopy (ms), fragmentation (msms), charge and electronicproperties (ms, IM, etc.), shape (IM, drift, etc.), bond and vibrationalproperties (various spectroscopic methods), and it's IROA form (basemass and number of carbons).

A Matrix is a standard well-defined Basic IROA mixture of compounds suchas metabolites, including anabolite and catabolite molecules, or othercompounds utilized or present in a given study and contains at least onecompound a pair of stable isotopes of the same element that differ inmolecular weight (AMU) by at least one AMU. The two isotopes are presentin the molecules of that at least one compound in a predetermined ratiothat is other than the naturally occurring ratio of those isotopes.

Various Matrices exist, but each matrix supports a specific analyticalsystem, such as plasma, human biopsies, wheat, urine, etc. In addition,a plurality of Matrices can be prepared for the same specific analyticalsystem.

A library is a group of compounds known to be present in a Matrix.

An Internal Standard (IS) is a chemical mixture of compounds that canrepresent either the lighter or heavier set of IROA compounds such asmetabolites, subset thereof of a Matrix sample, or other compoundspresent or utilized in a Matrix sample of a given study, and is insertedexogenously into every sample that is to be analyzed. Like a Matrix, theIS is a standard well-defined mixture of compounds. The chemicalcompositions of both the Matrix and IS are ideally identical.

As used herein, predetermined first and second stable isotope amountsare preferably present in “inverted ratios” of each other such as thosediscussed immediately above in which the number of the numerator of thefirst ratio is the number of the denominator of the second ratio, andthe number of the denominator of the first ratio is the number of thenumerator of the second ratio.

Taking the above ratios of 95% and 5%, a first ratio would be 95/512C/13C in the C-12 medium, whereas the second, inverted ratio, would be5/95 12C/13C in the C-13 medium. It is to be understood that acontemplated set of ratios need not be 95/5 and 5/95, and although thoseamounts are particularly preferred, they are used herein forconvenience.

It is to be understood that the first and second stable isotopes presentin a Matrix or any other exogenously provided composition such as aninternal standard are predetermined and as are their respective amountsof each isotope. As a consequence, the words “predetermined” and“stable” are rarely used herein with their presence implied to minimizeverbosity.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

A first aspect of this invention contemplates an IROA Matrix compositionof biologically-produced metabolites, including anabolite and catabolitemolecules, that is typically a room temperature solid that isdispersible or soluble in an aqueous medium (as defined hereinafter).The individual metabolites have a molecular weight of less than about2000 AMU, preferably about 1500 AMU or less, and more preferably lessthan about 1000 AMU. The lower weight limit for a contemplatedmetabolite is about 60-75 AMU as in acetic acid and glycine.

Every compound is equally present at both of two predeterminedisotopomeric balances such that each of the isotopomers is present atabout 2 to about 10% of isotope one and at about 90 to about 98% ofisotope two. Illustrative useful first and second isotopes of the sameatom are one or more elements that include the isotopes of carbon (12Cand 13C), nitrogen (14N and 15N), oxygen (16O, 17O, or 18O), sulfur(32S, 33S, 34S, or 36S), chlorine (35Cl and 37Cl), magnesium (24Mg, 25Mgand 26Mg), silicon (27Si, 28Si and 29Si), calcium (40Ca, 42Ca, 43Ca, and44Ca), and bromine (79Br and 81Br). The first and second isotopes arestable to radioactive decay (can be used in a laboratory without addedprotection from possible radiation injury), and are other than hydrogenand deuterium.

Put more explicitly in terms of the particularly preferred isotopes, C12and C13, one group of isotopomers contains about 2 to about 10% C13 andthe other group contains about 90 to about 98% C13. Preferably, a firstgroup contains about 5 to about 10% C13 and the second group containsabout 90 to about 95% C13, with the remaining carbon atoms being C12 ineach instance. It is particularly preferred that the first groupcontains about 5% C13 and the second group contains about 95% C13, withthe remaining carbon atoms being C12. This means that the IROA peakshape for each compound ideally is comprised as a perfectly balanced,symmetrical collection of peaks, with each half a mirror image of theother.

It is to be understood that the above-stated percentages are intended tobe identifiably different from the natural abundance amounts of the twoisotopes used. Thus, in the case of carbon isotopes, whose naturalabundances are 98.89% for C12 and 1.11% for C13, use of about 90 toabout 98% for one isotope and about 2 to about 10% of the other isotopepermits the analytical equipment to readily distinguish between naturalabundance peaks and those provided by an IROA Matrix. Use of the term“about” for the percentage of one or the other isotopomers present ismeant to be within ±3% of the stated amount. Thus, the above isotopepercentages are known and predetermined, but use of specific amountswithin the ranges stated is mostly a matter of convenience.

It is also to be understood that trace, impurity amounts of the elementused for an IROA study, here carbon, can also be present among the atomsof that element. Such trace amounts are typically of no consequence to astudy. For example, the Handbook of Chemistry and Physics, 54^(th) ed.,CRC Press, Cleveland, Ohio, page B251, 1973-1974, lists the naturalabundance of C12 and C13 as being 98.89 and 1.11 percents, respectively,with the presence of C14 being reported, but not its percent amount.

It is still further to be understood that use of the words “first” and“second” in regard to the isotopes and the several compositions that cancontain them is only for purposes of clarity to distinguish theisotopes, and is not meant to imply anything concerning the order ofcarrying out any manipulations.

It is preferred that IROA matrices be prepared in relatively largequantities, such as about 10 to about 100 g for an industrial scale andabout 10 to about 1000 mg on a laboratory scale so that each batch canbe utilized over many spectral analyses. The obtained Matrix compositionis preferably kept frozen such as at −80° C. until used to maximize itschemical stability and analytical reproducibility.

Another contemplated aspect of the invention is a method of creating areference library of identity data of compounds in an IROA Matrix asdescribed above, and comprises the steps of 1) mass spectrallydetermining the identity of the compounds of an IROA Matrix that arewithin the resolution and sensitivity of the apparatus to provide itssymmetrical IROA peak pattern, and additionally determining one or moreof: a) the gas and/or liquid chromatographic properties of the compoundspresent, b) the collisional cross section of the compounds present, andc) the fragmentation pattern of the compounds present. The compoundidentity data so determined is maintained for use in identifying one ormore of the same compounds in a later-analyzed sample. The referencelibrary of identity data of compounds in an IROA Matrix is itself alsocontemplated. The use of one or both of compound collisional crosssections and fragmentation patterns are preferred in conjunction withmass spectral identification.

A further contemplated invention is a method of quantifying andidentifying compounds in a natural abundance sample using an InternalStandard that is of the same chemical composition as isotopomerscontaining the about 90 to about 98% of the heavier molecular weightisotope-containing compounds of an IROA Matrix composition and isinserted into that natural abundance sample. Each compound in theInternal Standard is itself identified in a before-described referencelibrary of identity data. It is preferred that the quantity of eachidentifiable compound of the natural abundance sample is determined, andmore preferably, the quantity of each natural abundance sample compoundis determined relative to the Internal Standard.

Another aspect of the invention contemplates a method of qualityassurance and/or a quality control on the operational constancy of amass spectral apparatus and associated ion mobility channel andchromatographic apparatus, when present. This method contemplatescarrying out a mass spectral analysis on multiple Matrix samples duringthe course of carrying out analyses of different samples, anddetermining whether the same sets of symmetric IROA mass spectral peaksare present in each analysis. Illustratively, a Matrix sample can byanalyzed before an experimental sample is analyzed, after anexperimental sample is analyzed, after the next experimental sample isanalyzed. Interpretation of these Matrix analyses is discussed elsewhereherein.

Use of the above technique permits the user to simultaneously validateand quantitate a compound that is present in a complex mixture withoutthe need for a prior baseline separation. This technique benefits fromthe fact that a collection of isotopomeric ions of any compound, e.g., aC-13 based Isotopic Ratio Outlier Analysis (IROA) peak, an IROA pooledpeak, or any other combination of isotopomeric forms of the samemolecule, down to and including the dual collection of a C-12monoisotopic isotopomer paired with the C-13 monoisotopic peak, or evenisotopomers based on isotopomers of other elements, such as nitrogen,oxygen, sulfur, or others, share the same collisional cross section(CCS). As a consequence, the isotopomer ions pass through an ionmobility (IM) channel, e.g., high-field asymmetric waveform ion mobilityspectrometry (FAIMS), differential mobility spectrometry (DMS),structures for lossless ion manipulation (SLIM), trapped ion mobilityspectrometry (TIMS), and other drift tube and/or ion mobilityspectrometric (IMS) technologies to emerge at the same time as the samecollection that entered when it exits, or in a predictable fashiontherefrom.

The entire such collection of ions can then be subjected to afragmentation, which yields fragment ions, all of which bear the samenumber of isotopomers as the original collection. The identity of theoriginal compound is confirmed by the fragmentation patterns resulting,and its acquired mobility information can contribute to thisconfirmation.

The absolute quantity of any compound can be determined by comparison ifthe quantity of any of the subsets of the collection is known. The useof a liquid chromatographic (LC) separation prior to the entrance of theion collection reduces the number of ion collections that enter the IMchannel at any given time, which can be helpful but is not needed. Thistechnique can be used in the quantitative analysis of extremely complexmixtures, for instance, a tissue, cell, biopsy, or biofluid, human ornon-human.

In such a case, an appropriate isotopomeric internal standard (IS), IROAor otherwise, that contains a multitude of the same compounds at a fixedor known concentration can be added to the biological material. Theresulting pooled mixture can be analyzed and quantitated with completeconfirmation of identities without the need for chromatographic baselineseparation of the material as current practice requires.

A preferred embodiment of this method can include the preparation of thebiological sample, addition of the isotopomeric mixture, separation ofthe pooled material; first by an high-performance LC (HPLC) separation,generally LC coupled to mass spectrometry (LC/MS), followed by an IMchannel, and finally fragmentation by MS/MS. Other separation methodssuch as gas chromatography (GC), supercritical fluid chromatography(SFC), capillary electrophoresis (CE), or similar, ornon-chromatographic systems such as Solid Phase Extraction (SPE) oron-line methods can also be used to help but are not needed. Anycompound that is present in the IS can be quantitated at the level ofthe MS, or MS/MS. Identity is confirmed by MS/MS. See, e.g., Stupp, etal., Anal. Chem. 2013, 85 (24), 11858-11865.

Another aspect of the contemplated invention provides a new aspect tothe previously discussed Phenotypic IROA Protocol. This aspectcontemplates a reagent pair that is capable of transforming thebiologically-produced metabolite compounds of a natural abundance massspectral analysis sample into an IROA sample. This reagent paircomprises two reactively identical reagents that constitute first andsecond isotopomers.

The first isotopomers contain about 2 to about 10% of a first isotope,and second isotopomers contain about 90 to about 98% of a second isotopeof the same atom. The first and second isotopes are stable toradioactive decay and are other than hydrogen and deuterium.

It is preferred that the reagent molecule contain 4 or more atoms thatcan be one or the other of the isotopes of choice. The upper limit ofsuch atoms is typically a matter of convenience, with reagents that cancontain 6 to 10 atoms of possibly variant isotopes of choice beingpreferred.

Each of the reagent pair contains the same reactive group that reactswith and bonds to a functional group of one or more compounds present ina composition of biologically-produced natural abundance metabolitecompounds. Each of those metabolite compounds has a molecular weight ofabout 2000 AMU or less, preferably about 1500 AMU or less, and morepreferably about 1000 AMU or less.

It is noted that the phrase “reactive group that reacts with and bondsto a functional group” is not chemically accurate in that once reactedwith each other, the reactive group and the functional group are nolonger in existence so they cannot bond to each other. Rather it isresidues of each group that bond to each other. The latter phrase isthought to be cumbersome and therefore, the former, quoted, phrase isused with the understanding that the latter phrase more chemicallyaccurate is intended.

The reactive group of the reagent pair reacts with and bonds to afunctional group selected from the group consisting of one or more of anamine, aldehyde or ketone, hydroxyl, thiol and carboxylic acid. Thosereactive functionalities are present in proteinaceous metabolites andalso compounds containing sugars, as well as mostly oxidizedcarbonaceous condensation products such as the terpenoids such aslimonene, carvone and geraniol.

A particularly preferred reactive group reacts with and bonds to anamine group as is present as the amino-terminus of oligopeptides, aminoacids and compounds with exocyclic nitrogen atoms such as mescaline,serotonin, and dopamine.

One such particularly preferred reactive groups is an isothiocyanategroup. Isothiocyanate synthesis is well known in the art such that anisothiocyanato group containing a desired percentage of 13C can belinked to a carbonaceous group that itself can be prepared to contain adesired percentage of 13C so that desired pares of isotopomers can bereadily prepared. A particularly preferred isothiocyanate isphenylisothiocyanate (PITC).

In another preferred reagent pair, the reactive group reacts with andbonds to a ketone or aldehyde group. Here, reactive group is a hydrazineor a semicarbazine that forms a hydrazone or semicarbazide with a ketoneor aldehyde of a metabolite. Syntheses of these reactivegroup-containing compounds is also well known so that they too can belinked to carbonaceous moieties that contain a desired amount of 13C.

The Problem and Problem Solved

It is possible to use metabolomic techniques, such as the IROA basic, orIROA phenotypic protocols (optimally), or standard metabolomictechniques to identify and crudely quantify several hundred or eventhousands of compounds in a biological sample. However, until thepresent invention, in order to make such measurements and to compare themeasurements from any two or more samples, all the samples needed to beanalyzed in a single batch, ideally during a single day becauseday-to-day variances are too great to otherwise overcome, and absolutequantitation; i.e., relative to a known standard, cannot be assured.

It is currently not quantitatively acceptable to compare samples assayedon the same instrumentation several days apart, and impossible tocompare data generated on different instruments, or based on differentmethods. Instrument drift, chromatographic drift, and even environmentalconditions can alter results sufficiently so that reproducibility ishard to obtain even on the same instrument.

In addition to these problems of quantitation, the identification of anycompound across many mass spectral techniques alone is unlikely to besuccessful unless very careful calibrations have been made and authenticstandards are run. This is because, not only are there multiplebiological compounds that can be confused because they have the sameexact mass but, even more problematic, there are often more artefactualor fragmentary compounds that are structurally different from, but canshare the correct mass, or even formulae, as biological isobaricequivalents. The IROA workflow directly addresses these issues, andothers, on many levels and overcomes them.

The IROA workflow provides a “standard sample”, referred to as “theMatrix sample”, that is deeply analyzed multiple times during theanalytical session. The Matrix sample is randomly intermingled withexperimental or clinical samples. The identity and behavior of thecompounds in this Matrix sample are used to identify all of the samecompounds in the experimental samples based on their shared IROApatterns. There can be different Matrix sample types for differentanalytical situations; i.e., a “Matrix” for Blood plasma, a “Matrix” forhuman liver, or even a “Matrix” for wheat. The Matrix sample can containsynthetic IROA patterns in situations as described in IROA963, IROA964,and IROA251.

A Matrix sample is always constituted as the same carefully controlledmixture of compounds. Different compounds can be present at differentconcentrations; however in any given “Matrix” batch, each individualcompound is always present at the same concentration in all aliquotedMatrix samples. All the compounds present in the Matrix sample arehighly defined.

The Matrix sample is a Basic IROA sample, and thus every compound isequally present at both predetermined isotopomeric balances, such as thepreferred 5% C13 and 95% C13. This means that the IROA peak shape foreach compound ideally is comprised as a perfectly balanced, symmetricalcollection of peaks, with each half a mirror image of the other.

Because of the symmetry of the IROA peaks in the Matrix, Matrix samplescan be completely catalogued; even peaks deep into mass spectral noiseat extremely low levels well below what would otherwise be possible todiscern can be identified and characterized. The triple-redundancy ofthe Basic IROA peak guarantees the consistent interpretation, andidentification in every analysis.

Because all the compounds present in the Matrix samples can becatalogued, and because they are consistent in a given Matrix, theirchromatographic behavior, ionization efficiency, ion mobility (IM)characteristics, fragmentation behavior, and the like can be evaluatedand these values are used to correct for any day-to-day variances, whenthe analytical system is similar, or even if it is very dissimilar.

Because the majority of these compounds are found even across verydifferent analytical platforms; i.e., with different chromatographic,ionization, or detection systems, the IROA characteristics of the IROAprimary scan and the IROA secondary chemical characteristics, as seen inion mobility, SWATH [see, Gillet et al., Mol Cell Proteomics,11:011.016717 (Jun. 1, 2012)], or other fragmentation systems assurethat every compound in Matrix can be mapped from any analytical systemto any other analytical system, thereby providing a mechanism fordirectly comparing the complete Matrix chemical composition of any twomatrix samples, and through them any clinical or experimental samplesthey support.

A Table illustrating windowing widths for SWATH set to pass all of thedesired IROA compound peaks through them is shown below.

Max # carbons below center center window overlap count min max 3 59 10 51 49 69 4 74 15 8 2 59 89 5 89 15 8 3 74 104 7 119 30 15 4 89 149 9 14930 15 5 119 179 13 209 60 30 6 149 269 17 269 60 30 7 209 329 27 389 12060 8 269 509 36 509 120 60 9 389 629 54 749 240 120 10 509 989

Windowing schemes such as SWATH windows set as shown above permits allIROA peaks to passage through them to provide for the isolation of acomplete IROA peak set pattern as is shown in FIG. 12A. It is based onthe maximum number of carbons in any known metabolite with a mass of thecenter mass, and sets windows (minima and maxima), and overlapsaccordingly. FIG. 12B illustrates the phenylalanine fragmentationpattern that provides diagnostic structural information as seen fromFIG. 12C.

Because the chemical makeup and therefore chromatographic behavior ofthe Matrix sample is identical to the Internal Standard applied to theExperimental samples and analyzed within the same batch, it is possibleto use the in-depth, informationally-strong, triply redundant chemicalidentification information obtained from the Matrix sample and apply itto the Experimental samples.

The Matrix samples can be analyzed to find, identify, and collect allidentifying physical characteristics for all of the compounds containedwithin it with extreme accuracy and sensitivity. For every triplyredundant IROA peak, the physical information can include but is notlimited to information from the primary ms scans:

the retention time (RT), 12C monoisotopic mass, 13C monoisotopic mass,number of carbons contained in the molecule;

in-source and post-source fragmentation characteristics;

any physical characteristics gleaned from other methods applied to theeffluent stream, for instance, IR, UV;

various post source fragmentation methodologies, including for instance,collision-induced dissociation (CID), electron-capture dissociation(ECD), SWATH, etc., whether Directed (data dependent acquisition—DDA),Independent (data independent acquisition (DIA), such as MSe, SWATH,etc., ion mobility (IM);

or any other technique that can provide information to support theidentification of this IROA peak.

The experimental or clinical samples are biochemically complex andcontain a diverse assortment of compounds; however, the carbon isotopicbalance for these compounds is present only at natural abundance C13levels; i.e., approximately 1.1% C1.

An internal standard (IS) that is identical in concentration andchemical composition to the 95% C13 (or other suitable) isotopomericportion of the Matrix samples is added to each clinical sample. Thisaddition means that each experimental sample can be analyzed as aPhenotypic IROA sample because it now conforms to the Phenotypic IROAprotocol.

Because the same C13 isotopomeric IROA signal is present in both theMatrix and Experimental samples, and the chromatography is consistentacross both, the chemical compound identification and physicalcharacteristics seen, and verified, in the Matrix can be mapped directlyto the experimental samples. Because of the uniqueness of the IROAsignal in the IS placed into a redundant Phenotypic sample, the mappingdoes not require that the experimental samples also have the secondaryphysical characteristics, but rather the user can infer those secondaryphysical characteristics based on reference to a co-incidentallyanalyzed Matrix sample.

The Matrix and experimental samples are randomly interspersed into asingle sample set (for instance, such that there is one Matrix injectionfor every approximately 10 experimental injections), and the entiresample-set analyzed.

Because the samples have been completely and randomly intermixed duringthe analysis, the catalog of all peak pairs, their RT, number ofcarbons, IM and fragmentation characteristics provide information whereeach of these same IROA peaks is found in the experimental samples. TheNatural abundance peak is easily located and quantitated as itcollocates with it's IROA peak at a mass that is the mass of the IROA¹³C monoisotopic peak less the number of carbons it contains times themass of a neutron.

Quality Control, reproducibility, and accuracy for all samples analyzedaccording to the IROA workflow are assured because:

the Matrix sample is a “standard” sample, that is always the same, thecatalog of all IROA peaks found in each daily Matrix analysis provides away to quantitate the performance characteristics for theinstrumentation for every day's analysis and provides a mechanism forcorrecting any instrumental error or determining that the error on agiven day was un-acceptable;

the amount of IS introduced to every sample is identical to that in theMatrix and is the same across all samples, the sum of all signals in theIS is a constant and can be used to normalize samples if they are nototherwise normalized.

With the inclusion of an orthogonal, second-stage analysis and thecollection of data detailing additional physical characteristics, suchas an ion mobility, fragmentation, such as SWATH, UV, or IR, etc., thecompounds found in two sets of Matrix samples that have been analyzedunder very different analytical conditions can be unequivocally mappedfrom one to the other and therefore provide for the quantitativecomparison of the clinical or experimental samples associated with theirrespective Matrix samples.

This workflow can be automated in its entirety due to thetriple-redundancy of the compounds in Matrix samples, and the redundancyand equivalency of the clinical samples.

Thus, the IROA workflow combines the strengths of two IROA-basedprotocols to 1) provide a method for the quantitation of a very largenumber of compounds to be measured in a single analytical run, 2)provide a mechanism to correct any errors in quantitation irrespectiveof the analytical systems used, and 3) provide a mechanism to assurethat the identification of all compounds is consistent across time andanalytical platforms.

The Phenotypic IROA Protocol

The Phenotypic IROA protocol is a protocol for situations in which it isnot feasible or practical to label the experimental sample itself but acommon and consistent 95% (+/−3%) IROA internal standard, such as theabove described C13-IS, is used to assure accurate identification of amolecule and accurate quantitation. The Phenotypic Protocol is usefulfor the analysis of human (clinical) samples, agricultural samples,industrial samples, or other situations where the size or the source ofthe experimental samples is such that it is simply not feasible to labelthem. However, the Phenotypic protocol, by providing a common rigorousIROA internal standard, provides a more accurate route for theidentification and quantification of a large number of compounds thatare found in the sample natural abundance isolates.

Unlike the “unbiased” or “non-targeted” analysis of basic IROA,Phenotypic IROA is a targeted quantitative analysis of a very largenumber of compounds based on a very chemically complex IROA internalstandard (IS). A C13-IS can contain well over 1000 compounds(potentially unlimited), but the IROA properties outlined earlier do notrequire complete chromatographic separation to assure both the identityand quantitation of all the compounds contained in the IS.

The Phenotypic protocol puts an IROA internal standard into everynatural abundance sample and uses the dual pieces of information fromthe C13-IS, 13C-monoisotopic mass and number of carbons, to locate thenatural-abundance isotopomer of the same compound. Correlation of thenatural abundance time-resolved chromatographic profile of the foundpeak, and it's natural-abundance isotopic form are then used to supportthe IROA-based identification.

Because the IROA peaks are informatically self-contained, it is possibleto correctly identify and quantify multiple co-eluting peaks. In thecase of the Phenotypic Protocol, the IS can be created by a worker toprovide support for the unique quantitation needs of the experimentalsystem. Thus, a wheat researcher, can create a wheat C13-IS that can beused because it contains a chemical profile more reflective of wheatbiochemistry, but this C13-IS is used primarily to find and identifyIROA peaks in wheat and quantify their natural abundance counterparts.Although the triple redundancy of the Basic IROA protocol does not existin the Phenotypic protocol, the signal is still redundant in that the95% C13-IS provides a mass and number of carbons to determine exactlywhere the natural abundance monoisotopic signal is found (see FIGS. 5and 6).

In the IROA workflow the same C13-IS is used in both the Matrix and theClinical or experimental samples and the chemical information derivedfrom the Matrix sample is used to verify and validate the compoundsfound in the clinical or experimental (Phenotypic) samples. ThePhenotypic samples can be analyzed for chemical information to the sameextent as the Matrix samples but this is not required. For instance,whereas the Matrix samples need to be analyzed to completelycharacterize every compound present in it, it can be sufficient to usethe mass and retention information derived from the analysis of theMatrix to find the same compounds in the experimental or clinicalsamples, and use a higher acquisition rate than would be possible in theMatrix samples to achieve a higher quantitative accuracy.

The IROA Workflow

The IROA workflow combines and leverages the strengths of two previousIROA protocols, Basic IROA and Phenotypic IROA, and adds additionalabilities to resolve chemical identity, normalize data, and enhancereproducibility between samples and across platforms whether similar ordissimilar.

IROA workflow makes the best use of the Basic IROA signal to catalog,validate, and characterize all of the compounds in the Matrix and thusC13-IS (which is common, and consistent to both the Basic and Phenotypicsamples). By using the same C13-IS in the Phenotypic clinical orexperimental samples, all the chemical identification and validation ofthe Matrix sample, a Basic IROA sample, can be applied to theexperimental or clinical samples, which are Phenotypic samples.

In addition, the IROA Workflow applies an additional orthogonalidentification second stage, such as Ion mobility, in-source or postsource fragmentation, UV, IR, or the collection of other chemicalcharacteristics, for each IROA peak in the Matrix, to provide additionalunique physical attributes for every compound in the Matrix sample. Ifevery compound in the Matrix is uniquely identified and is mappable toevery clinical or experimental sample, this system supports completelyreproducible compound identification irrespective of the analyticalplatform.

Therefore, the C13-IS in the experimental samples is capable of bothproviding a complete identification and quantitation solution withoutthe need for a base-line chromatographic solution, and without the needfor using the same orthogonal identification system on these samples.This is of import because the secondary systems can lower the temporalresolution and thereby lower the precision of the analyticalmeasurement, but the measurement in Matrix is required for the mappingof chemical attributes and for identification purposes. Thequantification of the Matrix is needed only at a lower level ofprecision.

On the other hand, for the clinical or experimental samples thequantification precision should be as high as possible. This overallsolution has:

1) very high-level accuracy in identification and quantitation ofcompounds found in in the experimental sample due to the presence of theIS and mapped to the Corresponding compounds in Matrix,

2) a highly accurate and precise identification of all of the compoundsin the Matrix samples, and

3) a rich and continuous quality assurance/quality control (QA/QC) forall instrumentation parameters (again derived from the Matrix sample)that is applied to the clinical or experimental sample, which isrequired for making human-relevant clinical biochemical measurements.

The chemical identification of the compounds in each Matrix injectionderived from the secondary analytical streams, such as ion mobility,fragmentation, or other UV/vis, etc. provides sufficientcharacterization so that each compound can be uniquely identified basedon these secondary features. Therefore, the combination of IROA patternplus this secondary data, much of which is also IROA-based, provides amethod to provide the reproducibility (quantitative and qualitative)needed to compare samples across wildly differing analytical platforms,or to adjust for day-to-day variances of instrumentation, and to assureboth compound identity and quantification across differing platforms.

This workflow uses aspects of the Basic IROA protocol and a consistentMatrix sample to provide a (new) (QA/QC) that is independent of theinstrument or the chromatographic systems.

When the benefits of each of these two protocols are combined into asingle protocol they bring the strength of the triply-redundant BasicIROA protocol to build targeted libraries from highly standardized“Matrix” samples that are then used in the doubly redundant Phenotypicanalysis of clinical samples.

The Matrix and clinical samples both contain the same concentration ofthe same 95% C13-IS. Because the Matrix samples additionally contain amatched C12-B that has the same chemical profile, their combinedisotopic signals are symmetrical, mathematically balanced andunambiguously found. These libraries (catalogs of all compounds found)are created in Basic IROA samples at a higher level of stringency can beused, enabling a much broader assortment of compounds to be found evendeep in mass spectral noise. Because the Phenotypic samples rely on thesame 95% C13 internal standard, these libraries can be applied tocoincidentally run samples with perfect matching expected and can becompared to non-coincidental samples through their common Matrixreferences.

The novelty of this approach derives from a previously mentionedattribute of the IROA peaks, namely that all of the isotopomers of aparticular compound will share virtually identical chemical physicalattributes except for mass, including UV, and to a limited extent theIR, as the additional neutrons have little influence on the electronicfields, charge distributions, or electron configurations. Thus, the IROApeaks will co-chromatograph and be seen as IROA peaks in the MS scans,and will also move through both ion mobility, SWATH fragmentations, aswell as other processes, as complete units. The ability to find thembeyond the MS level is a novel observation in this regard that permitsthe IROA workflow to use these IROA attributes to qualify and interpretIROA peaks at all stages of the analytical and identification processand make the IROA workflow possible.

Matrix and the C13-IS are always the same chemical mixture, at the sameconcentration, therefore these libraries provide a basis for a newcross-platform, cross-instrument, time independent QA/QC that makes itpossible compare samples prepared in different laboratories, usingdifferent methods. The Matrix and C13-IS can be either biologically orchemically produced.

The Identification of Matrix Compounds

Each Matrix has a library associated with it when it is first prepared.The libraries are the compounds that can be seen reproducibly when aMatrix sample is chromatographically separated and the Basic IROA peaksin it are examined. Given the extreme diversity of possible chemicalstructures, the mass spectral data generated from chromatographicseparation alone is not sufficient to identify most compounds, and isnot even sufficient to identify a unique molecular formula for mostmolecules.

The Basic IROA peaks add to the monoisotopic mass the exact number ofcarbons in the molecule, and for most utilized libraries such asmetabolite libraries this is sufficient to provide a unique molecularformula. However, for many molecular formulae, a given formula can beshared by a large number of compounds, hence, although IROA provides anassured formula it does not, in and of itself provide assuredidentification.

The IROA Workflow analyzes Matrix on a regular basis. In addition to themolecular formula for each IROA peak, if we can add collisionalcross-section (CCS from IM), fragmentation data (ms/ms from SWATH orother techniques), UV, IR or any other physical characteristic of eachcompound in the Matrix and the library of compounds known to becontained in it, then the combination of assured molecular formula andthese physical attributes become unique identifiers for each compound.

The IROA workflow analyzes the Matrix sample to determine thechromatographic behavior of all library compounds in the Matrix and ISon a daily basis. Because the concentration of the compounds in Matrixand IS, and their chromatographic behavior are identical, anyidentification made in Matrix can be mapped to IS. The key to the use ofMatrix is that the clear IROA-formatted peaks maintain their integritythrough msms where all fragments will show as IROA fragmentation, andsimilarly through IM where all the IROA peaks share a common CCS.

Illustrative Preparation of Matrix and IS

Whereas a Matrix (and IS) can be created to be a perfect match to anysample, most living things share a common core metabolism and thereforea “generic” matrix can be produced that is suitable for identifying andquantifying a wide variety of compounds in a wide variety of sampletypes. For instance, almost all living things use the same 20 aminoacids and the same nucleotides, and share most of the same biochemicalpaths. Therefore, for good economic reasons one can opt to not create aspecific Matrix (and IS) for a given sample type, but rather use ageneric Matrix (and IS). A reasonable Matrix (and IS) can be createdfrom single-celled or multi-celled organisms. Single-celled organismssuch as fermentable yeasts, bacteria or alga where the efficiency of thefermentation process can be carefully controlled are preferred.

The preparation of a particularly preferred Matrix (and IS) isillustrated here, but a similar process could be followed to create amore specific Matrix:

1) A strain of S. cerevisiae that grows well on minimal media, such asS288C, or similar, is biochemically most competent, is selected and istested to assure that it grows on a 95% 13C U-glucose as a main carbonsource. If it passes this test and approximates normal growth habit tothe eye of a fermentation expert it is deemed suitable;

2) The selected strain can be serially gown in sequentially largercontainers until enough cells are available to initiate a large-scalefermentation, as for instance, a 20 Liter fermenter or larger. The mediafor these early fermentations is isotopically enriched glucose, withadded minerals, including a nitrate or other nitrogen source, andvitamins. When sufficient cells are achieved, such as a 50 ml late-stagegrowth, this material is transferred into a 20 L fermenter in which themain carbon source for growth is isotopically labeled glucose, a nitrateor other nitrogen source, minerals and vitamins. The fermenter isaerobically sparged with carbon-dioxide free air for the duration of thefermentation to lower ethanol production and assure that the only CO₂available is that produced from the isotopically labeled glucose. Duringthe fermentation, additional isotopically labeled glucose iscontinuously added to replace that consumed. Throughout the course ofthe fermentation aliquots of fermentation fluids are removed foranalysis, the cell density is determined, and media chemistrycontrolled. When the fermentation achieves an optimal density, latelog-phase growth, but before it proceeds to senescence, the entire cellmass is harvested. The filtered cellular (yeast) mass is recovered fromthe media and frozen at −80° C.

3) The frozen yeast mass is removed from the freezer, resuspended indoubly distilled ion free water, and extruded through a French press, orother method of cellular disruption, at least three times; i.e., untilit appears that substantially all of the yeast cells are ruptured. Thisruptured lysate is permitted to autolyze; i.e, be digested by its ownenzymes, for 24 hours at 45° C. to form a “yeast extract preparation”.

4) The solid portions of the resulting yeast extract preparation arecentrifugally separated as one fraction. The supernatant is filtered toremove fine particles, and then it is lyophilized as “yeast extract”.The resulting yeast extract is a very rich biochemical mixturecontaining most of the stable biochemicals and their intermediates.Because this is meant to be a generic extract, the presence of unstablebiochemical intermediates is minimized. If these relatively unstablebiochemicals, such as ATP, etc., are sought then a specialized Matrix isneeded.

For a Matrix, the above process is run to produce a 95% yeast extract(produced from yeast grown with 95% 13C U-glucose as a main carbonsource) and, in a separate run, to produce a 5% yeast extract (producedfrom yeast grown with 5% 13C U-glucose as a main carbon source). The 95%yeast extract is used as the C13 half of the Matrix, and the Matrix isalso the IS; i.e., these two compositions are obtained from exactly thesame, homogeneous material in order to be chemically identical. (Note:they are both present in exactly the same concentration (20 mg per 40ml) in both the Matrix and experimental samples. The 5% yeast extract(5% YE) is added in an equal proportion to the 95% yeast extract (95%YE) to provide the Matrix. Therefore, the Matrix, on mass spectralanalysis, provides perfectly symmetrical sets of peaks. The InternalStandard is identical chemically and at its components are present atthe same concentration as those in the Matrix, but the IS contains only5% C12 material. The addition of the experimental sample provides thesource of the C12 material which is to be measured.

The method for making up the Matrix and IS are as follows.

1) Weigh out an exact quantity of 95% YE, for instance, 90 mg. This isdissolved to create a 10 mg per ml solution by the addition of theappropriate amount of 50/50 water/ethanol, for this example exactly 9 mlof 95% YE is made.

2) Weigh out an exact quantity of 5% YE, for instance, 30 mg. This isdissolved to create a 10 mg per ml solution by the addition of theappropriate amount of 50/50 water/ethanol, for this example exactly 3 mlof 5% YE are made.

3) To make Matrix add equal volumes of the 95% YE solution, and the 5%YE solution. Thus, in this example add 3 ml of 95% YE to 3 ml of 5% YE,to form Matrix precursor.

-   -   a) Aliquot 4 ml of the Matrix precursor into each injection        vial, dry and seal under nitrogen and store at −80° C.

4) To make IS aliquot 50 μl of the 95% YE into a 2 ml vial, dry and sealunder nitrogen, and store at −80° C.

In the case of Matrix, the dried Matrix injection vial contains 20 μl of95% YE and 20 μl of 5% YE. When it is dissolved for injection, forinstance by addition of 40 μl of dH2O, and mass spectrally analyzed, theresulting spectrum has very symmetrical, and very identifiable IROApeaks.

In the case of IS, the 2 ml vial contains 0.5 mg (or 0.500 mg) of the95% YE. When this is dissolved in 1.2 ml (1200 μl) and 40 μl of thissolution is added to a dried prepared experimental sample, eachresulting experimental sample contains 20 mg of 95% YE, the same amountas is in the Matrix. The experimental samples will also contain the samecompounds as the Matrix and IS, but they are present at naturalabundance C13 levels, approximately 1.1%. Because mass spectral analysisof Matrix provided the identity of all of the Matrix compounds and thusthose of the IS, the exact placement of the natural abundance peak isknown for every compound. The height or area of the natural abundancecompound is measured with complete knowledge of its identity andrelative to the standard quantity of it 95% isotopomer.

Illustrated Case 1

Blood Work-Up in a Hospital/Clinical Lab

In the last 10 years mass spectrometry has moved forcefully into thefield of clinical measurements because the flexibility, sensitivity, andcost are generally more favorable than traditional methods. However, inorder to make a measurement, it is usually required to use an internalstandard and to get a clean baseline separation between the compound(s)to be measured and other compounds that could affect the ionizationefficiency, or cause confusion by accidently appearing where they arenot expected, namely during the measurement period.

The reasons for this are simple. The internal standard is criticalbecause the mass spectral ion source is potentially variable, the massspectral signals are sensitive to tuning, ion suppression, solventvariability, or even atmospheric conditions. An internal standard thathas a single peak, such as almost all non-IROA standards, can have itssingle peak confused with, or contaminated with an artefact that has avery similar mass.

When measuring natural abundance analytes this risk is normallymitigated by the dual approach of a) separating the analyte from allother compounds chromatographically and b) including an internalstandard. As long as the internal standard co-chromatographs with theanalyte and it is separated from potential confounders, the risk of afalse measurement is considered low enough to accept the result.Although these steps can lower the risk to an acceptable level when asingle compound is being measured, the risk is multiplied enormouslywhen hundreds of compounds need to be measured simultaneously, andbaseline separation cannot be assured.

A more secure system is thus needed:

if an internal standard (IS) bore one or more unique identifyingcharacteristics, that could assure it was the internal standard for aparticular compound and it could not be mistaken for an artefact;

if the IS co-chromatographed with the target compound, it would sufferall the source, and analytical variance of the target compound and wouldprovide a perfect point of quantitative comparison; and

if the IS co-chromatographed, shared all analytical variance, and couldbe uniquely matched to its target, a clean base-line separation would nolonger be required to make a good analytical measurement.

The IROA C13-IS meets all of these criteria, 1) for each compound theshape of the IROA cluster is determined completely by its formula, 2)the compounds in the C13-IS co-chromatograph with their naturalabundance (in the case of clinical or experimental samples) or theirC12-B (in the case of matrix) isotopomers, and 3) are chemicallyotherwise identical.

Discovery Example 1

Illustratively, assume that the C13-1S and the Matrix are distributed asdried powder. The C13-IS is a dry powder in a 2 ml vial containing 500μg C13-IS, and the Matrix is a dried powder in a glass injection vialwith a glass insert that contains 20 μg C13-IS and 20 μg C12-B.

Further assume that there are 50 plasma samples to be analyzed, using astandard plasma preparation protocol such as:

50 μL of each of 50 samples of plasma are put into 50 1.8 ml Eppendorftubes. These constitute the 50 samples to be analyzed;

addition of 400 μL of cold precipitation solution (8:1:1acetonitrile:methanol:acetone) with repeater pipette to make a solutionof 1:8 (sample:solvent) ratio;

vortex sample to ensure mixing, cool sample @ 4° C. for 30 minutes tofurther precipitate proteins; centrifuge at 20,000 rcf for 10 minutes at<10° C. to create a pellet of proteins; transfer 375 μL of supernatantto new, labeled tube making sure to leave behind protein pellet;

dry the liquid sample using nitrogen, argon or other gas inert toreaction under the utilized conditions, in an Organomation AssociatesMultiVap® or similar apparatus, and store dried capped samples @ −80°until ready to reconstitute.

Assume that the C13-1S and the Matrix are distributed as dried powder.The C13-IS is a dry powder in a 2 ml vial containing 500 μg C13-IS, andthe Matrix is a dried powder in a glass injection vial with a glassinsert that contains 20 μg C13-IS and 20 μg C12-B.

The C13-IS is reconstituted by addition of 1.25 ml cold 80% aqueousmethanol; i.e., 20 μg per 50 μl, and vortexed to ensure mixing, thenallowed to rest @ 4° C. for 10 minutes.

50 μl of the reconstituted C13-IS is used to reconstitute each driedcapped sample. The sample is vortexed, allowed to rest 1 minute, and 40μl is transferred to a glass injection vial with a glass insert.

The Matrix is reconstituted in 50 μl of 80% aqueous methanol, vortexed,and allowed to rest 1 minute.

The Matrix and 50 experimental samples are transferred to a massspectrometer for chromatographic separation and MS analysis. All sampleinjections will be 4 μl injections, and thus contain the sameconcentration of 013-IS. The Matrix sample will be injected at least 5times, randomly within the sequence of the experimental sample.

The data sets from these analyses are analyzed as follows:

Software such as the ClusterFinder™ software (IROA Technologies LLC, AnnArbor, Mich.) can be used to find and characterize all of the IROA peaksthat can be found across the multiple matrix injections. It accumulatesall associated identifying characteristics for all IROA peaks found,including retention time (RT), C12 monoisotopic mass, C13 monoisotopicmass, number of carbons in the molecule, Ion mobility characteristics,fragmentation characteristics (in source, and post source), theamplitude of each peak in every IROA peak, the relationships between allIROA peaks, and any additional physical characteristics that wererecorded. The software uses all of the information found to identifyeach peak. Most peaks are well-known and previously well characterized,but possibly the software needs to create a new identifier. Themolecular formula for each peak is derived from its IROAcharacteristics.

The software provides a file (typically written) that summarizes itsfinding with regard to each compound found. It can include RT (averageand range, start of peak to end of peak), C12 monoisotopic mass (averageand range), C13 monoisotopic mass (average and range), formula, andidentity. On the standard high-resolution instruments available today,such as those made by Agilent, Thermo-Fisher, Sciex, and the like, theranges found are quite tight.

This file is the basis of a targeted analysis of each experimental file,such that for every compound found in the Matrix sample, a detailedtargeted analysis is run. Because the C13-IS is the same in every sampleit is found in every sample. Because the mass of its natural abundanceC13 monoisotopic is known it is possible to accurately quantify itspresence.

If the C13-IS is seen but no natural abundance is seen, it can belabeled as absent.

If a C13-IS is absent but was seen in matrix, it can indicate a qualitycontrol issue.

The sum of all peaks within either the C13-IS and the natural abundanceclusters is the numeric output.

Because the absolute amount of C13-IS in each vial is identical, the sumof all C13-IS peaks in each vial is fairly close if not identical.Deviations in this sum indicate problems either in the injection (ifonly one file shows it, or in the instrument if it shows a trend.

If normalization is needed, the assumption can be made that the sum ofarea on the natural abundance side is approximately equal, and if theC13-IS sums are relatively constant, then the natural abundance sum canbe normalized to the C13-IS sum. (Note: This is only be needed inextreme cases.)

The outcome of this analysis is a standardized measure of all of thecompounds present in the experimental (or clinical) sample. For eachsample there is an associated quality measure. The standardization to aconsistent Matrix sample assures nomenclature, within the sample set,across days within similar instruments, or even across widely divergentinstruments, although the performance differences across differentinstruments will likely cause some consistent differences that can bewell characterized due to the instruments source, lensing, detectors,etc. Nonetheless, it is expected that the majority of compounds will bemappable across widely differing platforms.

Illustrated Case 2

Urine Work-Up in a Hospital/Clinical Lab

Although there are some similarities, the chemistry of urine is verydifferent from that of plasma so urine is used to illustrate a variationof the IROA Workflow one in which the Matrix is custom-made for urine.

Consider the following as one possible variation:

500 (or more) liters of urine are acquired and dried. The powderednatural abundance urine is then split into two aliquots one thatrepresents 90% of the total amount obtained (aliquot A), and the otherthat represents 10% of that total amount (aliquot B). Each aliquot isderivatized using an IROA-based derivatization reagent, such asphenylisothiocyanate (PITC), although many others could be used. Thereare seven carbons in the resulting phenylthiocarbamyl reaction product,so if derivatization is done using both a 5% ¹³C PITC and a 95% ¹³CPITC, the resulting products can be mixed together to yield seven carbonIROA peak patterns.

In a similar manner, 5% ¹³C phenylhydrazine and a 95% ¹³Cphenylhydrazine that each contain six carbon atoms can be used toderivatize aldehydes and ketones in a sample such as urine as discussedabove for PITC. In the presence of excess phenylhydrazine, some sugarsadd two phenylhydrazine groups to form a diphenylosazone.Phenylsemicarbazide that contains seven carbon atoms can similarly beused to react with ketones and aldehydes present in a sample to beanalyzed.

The above-discussed reagents react with and bond to primary amines andaldehydes/ketones, respectively. A worker of ordinary skill can readilyconsult common texts such as Green et al., Protective Groups in OrganicSynthesis, 3^(rd) ed., John Wiley & Sons, Inc. New York, 1999, orLundblad, Techniques in Protein Modification, CRC Press, Boca Raton,1995, to identify further reagents that can be used to convert otherfunctional groups such as hydroxyl groups, thiols and carboxylic acidspresent in compounds of an all natural abundance sample into an7 IROAsample as discussed herein.

Therefore, with a view to the amounts needed reacting aliquot A with 95%¹³C PITC and aliquot B with 5% ¹³C PITC yields two mixed products inwhich all amine-containing compounds in urine are converted to theirphenylthiocarbamyl equivalents. The material from aliquot A and aliquotB are mixed in equal quantities to form a Urine-specific Matrix, whereasthe additional material from aliquot A provides a comparable C13-IS whenadded to the experimental samples.

Once these reagents are at hand a protocol similar to that ofIllustrated case 1 can be followed to quantitate all of theamine-containing compounds in urine, where they are plentiful. Differentderivatization reagents can be used to highlight other chemicalfunctionalities. In fact, if the products are as stable and relativelynon-reactive to one another, two or more can be individually created andthen pooled to create a more complex Matrix.

Each of the patents, patent applications and articles cited herein isincorporated by reference. The use of the article “a” or “an” isintended to include one or more.

The foregoing description and the examples are intended as illustrativeand are not to be taken as limiting. Still other variations within thespirit and scope of this invention are possible and will readily presentthemselves to those skilled in the art.

What is claimed:
 1. An IROA Matrix composition of biologically-producedmetabolite compounds, each of said metabolite compounds having amolecular weight of about 2000 AMU or less, and each of said metabolitecompounds being present as first and second isotopomers that are equallypresent at two predetermined isotopomeric balances, said firstisotopomers containing about 2 to about 10% of a first isotope, and saidsecond isotopomers containing about 90 to about 98% of a second isotopeof the same atom, said first and second isotopes being stable toradioactive decay and other than hydrogen and deuterium.
 2. The IROAMatrix composition according to claim 1, wherein saidbiologically-produced metabolite compounds are obtained from a celllysate preparation obtained from culture of single-celled ormulti-celled organisms, and the molecules are randomly and universallylabeled.
 3. The IROA Matrix composition according to claim 2, whereinthe cells of said cell lysate preparation are obtained from culture ofsingle-celled organisms and the molecules are randomly and universallylabeled.
 4. The IROA Matrix composition according to claim 1, whereinsaid first and second isotopes of the same atom are one or more elementsselected from the group consisting of isotopes of carbon (12C and 13C),nitrogen (14N and 15N), oxygen (16O, 17O, or 18O), sulfur (32S, 33S,34S, or 36S), chlorine (35Cl and 37Cl), magnesium (24Mg, 25Mg and 26Mg),silicon (27Si, 28Si and 29Si), calcium (400a, 42Ca, 43Ca, and 44Ca), andbromine (79Br and 81Br).
 5. The IROA Matrix composition according toclaim 1, wherein said first and second isotopes of the same atom are 12Cand 13C.
 6. The IROA Matrix composition according to claim 1, whereineach of said metabolite compounds has a molecular weight of about 1500AMU or less.
 7. A method of creating a reference library of identitydata of compounds in an IROA Matrix of claim 1 that comprises the stepsof 1) mass spectrally determining the identity of the compounds of saidIROA Matrix that are within the resolution and sensitivity of theapparatus to provide its symmetrical IROA peak pattern, and additionallydetermining one or more of: a) the gas and/or liquid chromatographicproperties of the compounds present, b) the ion mobility of thecompounds present, and c) the IROA fragmentation pattern of thecompounds present, and 2) maintaining the compound identity data sodetermined for use in identifying one or more of the same compounds in alater-analyzed sample.
 8. The method according to claim 7, wherein theidentity of compounds of said IROA Matrix are additionally determined bycollisional cross sections of the compounds.
 9. The method according toclaim 7, wherein the identity of compounds of said IROA Matrix areadditionally determined by the IROA fragmentation pattern of thecompounds present.
 10. The reference library of identity data ofcompounds in an IROA Matrix of claim
 7. 11. A method of quantifying andidentifying compounds in a natural abundance sample using an InternalStandard that is of the same chemical composition as isotopomerscontaining the about 90 to about 98% of the heavier molecular weightisotope-containing compounds of an IROA Matrix composition are insertedinto said natural abundance sample and the so combined sample isanalyzed at least by mass spectral analysis, wherein each compound insaid Internal Standard is itself identified in a reference library ofidentity data of claim
 10. 12. The method according to claim 11, whereinthe IROA characteristics of each compound in the Internal Standard areused to support the identity of each compound.
 13. The method accordingto claim 11, wherein the quantity of each natural abundance samplecompound is determined.
 14. The method according to claim 11, whereinthe quantity of each natural abundance sample compound is determinedrelative to said Internal Standard.
 15. A method of measuring qualityassurance and/or a quality control on the operational constancy of amass spectral apparatus and associated ion mobility channel andchromatographic apparatus, when present, that comprises the steps ofassaying the sample of an IROA Matrix composition of claim 1, anddetermining whether the same sets and amplitudes of symmetric IROA massspectral peaks are present in each analysis.
 16. The method ofdetermining quality assurance and/or a quality control according toclaim 15, wherein said biologically-produced metabolite compounds areobtained from a cell lysate preparation.
 17. The method of determiningquality assurance and/or a quality control according to claim 15,wherein the cells of said cell lysate preparation are obtained fromculture of single-celled or multi-celled organisms.
 18. The method ofdetermining quality assurance and/or a quality control according toclaim 15, wherein said first and second isotopes of the same atom areselected from the group consisting of isotopes of carbon (12C and 13C),nitrogen (14N and 15N), oxygen (16O, 17O, or 18O), sulfur (32S, 33S,34S, or 36S), chlorine (35Cl and 37Cl), magnesium (24Mg, 25Mg and 26Mg),silicon (27Si, 28Si and 29Si), calcium (40Ca, 42Ca, 43Ca, and 44Ca), andbromine (79Br and 81Br).
 19. The method of determining quality assuranceand/or a quality control according to claim 15, wherein said first andsecond isotopes of the same atom are 12C and 13C.
 20. The method ofdetermining quality assurance and/or a quality control according toclaim 15, wherein each of said metabolite compounds has a molecularweight of about 1500 AMU or less.
 21. A reagent pair capable oftransforming the biologically-produced metabolite compounds of a naturalabundance mass spectral analysis sample into an IROA sample thatcomprises two reactively identical reagents that constitute first andsecond isotopomers, said first isotopomers containing about 2 to about10% of a first isotope, and said second isotopomers containing about 90to about 98% of a second isotope of the same atom, said first and secondisotopes being stable to radioactive decay and being other than hydrogenand deuterium, each of said reagent pair containing the same reactivegroup that reacts with and bonds to a functional group of one or morecompounds present in a composition of biologically-produced metabolitecompounds, each of said metabolite compounds having a molecular weightof about 2000 AMU or less.
 22. The reagent pair according to claim 21,wherein said reactive group reacts with and bonds to a functional groupselected from the group consisting of one or more of an amine, aldehydeor ketone, hydroxyl, thiol and carboxylic acid.
 23. The reagent pairaccording to claim 22, wherein said reactive group reacts with and bondsto an amine functional group.
 24. The reagent pair according to claim23, wherein said reactive group is an isothiocyanate.
 25. The reagentpair according to claim 24, wherein said isothiocyanate isphenylisothiocyanate.
 26. The reagent pair according to claim 22,wherein said reactive group reacts with and bonds to a ketone oraldehyde group.
 27. The reagent pair according to claim 26, wherein saidreactive group is a hydrazine or a semicarbazide.